Based on the thermal gradient pcr mechanism, the temperature gradient determined by number of wells is important factor for determination of amplification optimization. So the used maximum gradient temperature range between 15 oC and 30 oC advances the speed and specificity of PCR. The thermal gradient also provides optimization for the temperatures of denaturation, annealing or extension in one experiment. It is also useful for many applications containing the cloning and sequencing in molecular biology. 
Touchdown pcr increases the specificity, sensitivity and yield of PCR cycles by using a special cycling program at which annealing temperature is decreased like 1-2 C for every second cycle. The initial annealing temperature is more than estimated Tm of the primers, while then it is decreased to calculated annealing temperature of the primers. This difference between the temperatures allows the exponential advantage of two-fold per cycle. It is useful in cDNA libraries and also the screening of single nucleotide polymorphism applications. 
Nested pcr is a method modified from standard PCR that avoids product contamination formed by amplification of undesired primer binding sites. In the nested pcr, primers are designed as two primer pairs including the one for outer fragment and one for the inner fragment such as outer forward – reverse primer and also inner forward – reverse primer. By this way, the large numbers of amplified outer fragments allow to continue the amplification of inner fragments resulting in the amplification with minimum contamination. 
Multiplex pcr technique depends on the combination of usage multiple primers and DNA polymerase enzyme for amplification of multiple targets DNA sequences in a single PCR thermal cycler. Multiple pcr performs the amplification of multiple targets by using single template or multiple templates. By this technique, the significant savings can be obtained in time with desired target DNA sequences. It is also used in many applications such as RNA detection, analysis of mutations and pathogen, etc. 
HOT START PCR
Hot start pcr technique allows decreasing non-specific amplification and also facilitates to set up the reaction at room temperature. In this technique, the ambient temperatures are used for preventing the polymerase activity which also activated at independent reaction temperatures resulting in the dissociation of it from its inhibitors and starts the polymerization. Hot start DNA polymerases are generally preferred for many applications needed a high degree of specificity. 
REVERSE TRANSCRIPTION PCR
If the starting material is RNA, the reverse transcription pcr is used by also helping of the reverse transcriptase enzyme. This enzyme transcribes the RNA from mRNA into cDNA used as template. This method is useful for many applications such as analysis of gene expression, identification of pathogen, etc. 
- Gradient PCR Machine
- Nature Protocols 3, 1452 – 1456 (2008)
Published online: 21 August 2008 | doi:10.1038/nprot.2008.133
Subject Category: Nucleic acid based molecular biology
- Nested Primers for PCR
Copyright 2002 Department of Biology, Davidson College, Davidson, NC 28036
- Multiplex PCR
- D’Aquila, R.T. et al. Maximizing sensitivity and specificity of PCR by pre-amplification heating. Nucleic Acids Res. 19, 3749 (1991).
Chou, Q., Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications. Nucleic Acids Res. 20, 1717-1723 (1992).
- Bustin S. (ed) (2004) A-Z of Quantitative PCR. IUL Biotechnology Series, International University Line, La Jolla, California.